Letter pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate Katsuhiko Mitachi,† Shou M. Kurosu,† Shakiba Eslamimehr,† Maddie R. Lemieux,† Yoshimasa Ishizaki,‡ William M. Clemons, Jr.,§ and Michio Kurosu*,†
Org. Lett. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/31/19. For personal use only.
†
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, Tennessee 38163, United States ‡ Laboratory of Microbiology, Institute of Microbial Chemistry (BIKAKEN), 3-14-23, Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan § Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Boulevard, Pasadena, California 91125, United States S Supporting Information *
ABSTRACT: We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.
N
-linked glycans play essential roles in many biological processes. Aberrant protein glycosylation is frequently observed in cancer cells. The integral membrane enzyme Nacetylglucosaminephosphotransferase 1 (DPAGT1) plays a central role in N-linked glycoprotein biosynthesis, catalyzing the first step in the dolichol-linked oligosaccharide pathway. Certain cancer cells require an increase in N-linked glycosylation for their progression, thus selective DPAGT1 inhibitors have therapeutic potential.1 The only inhibitors of DPAGT1 that have been reported to date are the antibiotic tunicamycin and its derivatives. Tunicamycins have been useful experimental tools to interrogate N-linked glycosylation and to examine the effects on protein folding.2 Tunicamycins inhibit growth across a wide range of mammalian cell lines in a nonselective fashion.1a In addition, physicochemical and biological properties of tunicamycins (e.g., water solubility and narrow therapeutic window) are far from ideal as leads for the development of preclinical drugs. Therefore, new types of inhibitors are required. We have recently identified a strong DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB, 1), by screening of an aminoribosyl uridine library generated based on muraymycin (Figure 1).1a,b APPB selectively inhibits growth of solid tumors (e.g., KB, LoVo, SK-OV-3, MDA-MB-432S, HCT116, Panc-1, and AsPC-1) at low micromolar concentrations but does not inhibit growth of healthy cells at these same concentrations.1a APPB displayed a remarkably selective cytotoxicity (e.g., IC50 pancreatic cancer cells/IC50 healthy cells: >1/100), metabolic stability (t1/2 > 60 min), and water solubility (∼75 mg/mL for HCl salt). Thus, pharmacological studies of APPB and its © XXXX American Chemical Society
Figure 1. Structures of anticancer DPAGT1 inhibitors, APPB (1), APPU (2), and tunicamycin.
related analogues using appropriate animal models are a focus of our continuing research efforts. APPB is a total synthetic product that cannot be accessed from reported natural products or muraymycin biosynthetic intermediates.3 Based Received: November 20, 2018
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DOI: 10.1021/acs.orglett.8b03716 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
transition-metal-mediated selective Boc protection in aqueous NaOH in a mixture of DMF, MeOH, and H2O. Because of the strong coordination aptitude of group 4 elements to diamines, we screened Cu, Ni, Zn, V, Co, and Fe salts for selectively synthesizing the mono-Boc-protected triamine 7 in excellent yield. The transition metal salts tested, selectivity of mono/diBoc (7/8), and isolation yield for 7 are summarized in Table 1.
on our structure−activity relationship studies of APPB, it was realized that a urea analogue, aminouridyl phenoxypiperidinbenzyl urea (APPU, 2 in Figure 1), shows biological activity equal to that of APPB without reducing the favorable physicochemical properties. Importantly, APPU has the potential to be synthesized from a biosynthetic intermediate of muraymycin (3) whose structure corresponds to de-Nmethyl FR-900463.1d,4 The intermediate 3 can potentially be produced at large scale by the disruption of the mur30 gene in the muraymycin-producing Streptomyces sp. NRRL 30471 (Figure 2).3 However, no synthetic method has been developed to convert the non-biologically active intermediate 3 to a pharmaceutically interesting product.5
Table 1. Selective Boc Protection of 4a
entry
MLx
time (h)
selectivity (7/8)c
yield for 7 (%)d
1 2 3 4 5 6 7 8 9 10 11 12 13 14
CuSO4 Cu(OAc)2 CuBr2 CuCl2 NiCl2·6H2O Ni(OAc)2·nH2O Ni(NO3)2·6H2O VCl3 Zn(NO3)2·6H2O ZnCl2 CoCl2 FeSO4·H2O FeCl3·6H2O
12 12(1)b 12 12(5)b,e 12(5)b 12(5)b 12(5)b 12 12(3)b 12(3)b 12(3)b 12(3)b 12(3)b 12(1)b
1:0 1:0 1:0 1:0 >19:1 >19:1 >19:1 4:1 1:1 1:1 1:1 1:1 1:1 1:1
20 95 10 90 85 85 85 40 45 40 45 40 40 35
Figure 2. Biosynthesis of muraymycins in Streptomyces sp. NRRL30471. a
Reaction condition: 3 (1.0 equiv), MLx (3.0 equiv), and NaOH (1 N, 4.0 equiv) in DMF−MeOH−H2O (1/1/1, 0.05M), after 30 min; (Boc)2O (2.5 equiv). bTime in the parentheses: time required for the reaction to be completed. cSelectivity was determined via HPLC analysis for the reaction mixtures after 12h. dIsolated yield. eSame reaction with CuCl2 (1.0 equiv) provided 6 in >90 yield in 1 h.
In a semisynthetic approach with 3, the most challenging transformation is the selective carbamation (temporary protection) of the primary amine (C5″-position) of the aminoribose moiety that allows functionalization at the other primary amine (C3′′′-position). In this communication, we report a semisynthesis of APPU (2) from 3 by exploring transition metals that temporarily protect the 2-((3aminopropyl)amino)acetamide system to achieve selective carbamation at the C5″-amine (Scheme 1). We have previously demonstrated amidation of protectinggroup-free amino acid derivatives with NH4Cl (excess), EDCI, glyceroacetonide-Oxyma, and NaHCO3 in water.1d,6 Under the same condition, 37 was converted to the corresponding amide 4 in over 95% yield (Scheme 1). The amide 4 was examined in
Reactions of 4 with a majority of the metal salts in Table 1 caused precipitation in a single solvent system (e.g., MeOH, H2O, or DMF); all reactions were carried out in a mixture of DMF−MeOH−H2O (1/1/1). Without addition of a transition metal salt, Boc protection of 4 furnished a 1:1 mixture of 7 and 8 (entry 14).8 Boc protection reactions in the presence of Zn salts, CoCl2, or Fe species provided a 1:1 ratio of 7/8 with 19:1 (entries 5−7). Selective Boc protection reactions mediated via the Cu species provided the only desired product 7 in 10−95% yield (entries 1−4). CuSO4- and CuBr2-mediated reactions did not progress toward completion even after 12 h (entries 1 and 3). On the other hand, selective Boc protection performed with Cu(OAc)2 or CuCl2 provided 7 in 90−95% yield (entries 2 and 4). Kinetically, the Cu(OAc)2-mediated reaction to the production of 7 is more favorable than that with CuCl2; the reaction was completed in 1 h with Cu(OAc)2 (vs 5 h with CuCl2). It may be attributed to the stronger coordination ability of CuCl2 to the C5″-amine, slowing down the electrophilic reaction with (Boc)2O. This hypothesis
Scheme 1. Semisynthetic Strategy of 1 from 2
B
DOI: 10.1021/acs.orglett.8b03716 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters was proven correct by the successful reaction in the strictly controlled condition (1.0 equiv of CuCl2); 7 was synthesized in >90% in 1 h. We also examined selective Cbz protection of 4 via the Cu(OAc)2-mediated condition developed in Table 1 (Scheme 2). In both cases (CbzCl and CbzOSu), 4 could be converted to the desired mono-Cbz-protected product 9 in 95% without formation of the di-Cbz byproduct 10. Scheme 2. Selective Cbz Protection of 4
Figure 3. Plausible 4−CuCl complex proposed based on NMR analyses.
Scheme 3. Synthesis of APPU (1) from 9 To obtain insight into mechanisms of the Cu-mediated selective Boc protection, we performed NMR studies for the Cu−4 complexes. Unfortunately, all Cu salts in Table 1 showed poor solubility in the complexation with 4 in conventional deuterated solvents (i.e., D2O, DMSO-d6, acetone-d6, and CD3OD). Thus, we synthesized two model compounds 11 and 12, and a mixture of 11 and 12 was applied to investigate complexation with the Cu salts in solution. Complexation of 11 and 12 with CuCl2 resulted in less precipitate and higher-resolution NMR. 1H NMR of a 1:1:1 mixture of 11, 12, and CuCl2 in D2O revealed that all carbon protons of the 2-((3-aminopropyl)amino)acetamide moiety of 11 are shifted to downfield; Δδ values were +0.73, +0.50, +0.26, and +0.05 for C2−H, C1′−2H, C2′−2H, and C3′-2H, respectively. In the 13C NMR of the 11−CuCl complex, no chemical shifts were observed due to the complexation of 11 with CuCl2. Although participation of the carbonyl oxygen in the 11−CuCl complex cannot be determined by these NMR experiments, coordination of the Cu-amide derivatives has previously been reported; based on X-ray and spectroscopic studies, the carbonyl groups of the primary amides are responsible for the formation of the stable Cu(II) complexes.9 In our NMR analyses, in the presence of CuCl2, the C5−2H in the aminoribose derivative 12 was not shifted (Δδ = 0), thus these data in the solution state may indicate that the C5″amino group in 4 remains reactive in electrophilic reactions. Considering these facts, we speculate that the coordination geometries around the Cu(II) ion and 2-((3-aminopropyl)amino)acetamide moiety (highlighted in red) of 4 are as shown in the 4−CuCl complex in Figure 3. Finally, we examined the urea formation of the mono-Bocprotected molecule 9 with the imidazole carboxamide derivative 13 and deprotection to synthesize APPU (2). The coupling reaction between 9 and 13 was best performed in a mixed solvent system of DMF−CH2Cl2 (1/1) in the presence of Et3N to furnish urea 14 in 90% yield. Deprotection of 14 with 30% TFA provided APPU·TFA (2−TFA). Purification of 2−TFA salt was performed with an ion-exchange column (DOWEX 50Wx4, H+) to afford 2 in quantitative yield. The synthetic APPU (2) in Scheme 3 was determined by 1H and 13 C NMR, MS, and HPLC to be identical to a sample previously synthesized via a total synthesis (see the Supporting Information).1d
In conclusion, we have established the semisynthesis of an anticancer agent, APPU (2), from the muraymycin biosynthetic intermediate 3. It was demonstrated that the 2-((3aminopropyl)amino)acetamide moiety in the amide derivative 4 forms complexes with Cu species either in water or in watercontaining organic solvent systems. Such complexes can serve as protection of the primary amine at the C3″-position in 4, allowing us to perform selective carbamate formation reactions of the primary amine at C5″-position. Synthetic pathways illustrated in Schemes 1 and 3 warrant a wide range of applications of the biosynthetic intermediate 3 for development of novel agents to cure human diseases (i.e., infectious and oncology fields). We are currently studying disruption of the mur30 gene of Streptomyces sp. NRRL 30471 to improve production of 3 (Figure 2).10 Practicality of isolation and purification of 3 with the genetically modified Streptomyces sp. and a larger-scale semisynthesis of a DPAGT1 inhibitor with strong anticancer activity will be reported elsewhere.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03716. Experimental procedures and copies of NMR (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Michio Kurosu: 0000-0003-0092-0619 C
DOI: 10.1021/acs.orglett.8b03716 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Institutes of Health (Grant No. GM114611). M.K. also thanks the University of Tennessee Health Science Center for generous financial support (UTHSC College of Pharmacy, CORNET, and UTRF awards). NMR data were obtained on instruments supported by the NIH Shared Instrumentation Grant. Streptomyces sp. NRRL 30471 was acquired from USDA (NRRL Culture Collection).
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REFERENCES
(1) (a) Kurosu, M. J. Mol. Pharm. Org. Process Res. 2018, 6, 141. (b) Mitachi, K.; Eslamimehr, S.; Kurosu, S. M.; Kurosu, M. Abstracts of Papers, 256th ACS National Meeting & Exposition, Boston, MA, 2018. MEDI-390. (c) Mitachi, K.; Aleiwi, B. A.; Schneider, C. M.; Siricilla, S.; Kurosu, M. J. Am. Chem. Soc. 2016, 138, 12975. (d) Mitachi, K.; Yun, H. G.; Kurosu, S. M.; Eslamimehr, S.; Lemieux, M. R.; Klaić, L.; Clemons, W. M.; Kurosu, M. ACS Omega 2018, 3, 1726. (2) (a) Takatsuki, A.; Arima, K.; Tamura, G. J. Antibiot. 1971, 24, 215. (b) Gao, Y.; Feng, H. C.; Walder, K.; Bolton, K.; Sunderland, T.; Bishara, N.; Quick, M.; Kantham, L.; Collier, G. R. FEBS Lett. 2004, 563, 185. (c) Yuste-Checa, P.; Vega, A. I.; Martin-Higueras, C.; Medrano, C.; Gamez, A.; Desviat, L. R.; Ugarte, M.; Perez-Cerda, C.; Perez, B. PLoS One 2017, 12, No. e0179456. (d) Hou, H.; Sun, H.; Lu, P.; Ge, C.; Zhang, L.; Li, H.; Zhao, F.; Tian, H.; Zhang, L.; Chen, T.; Yao, M.; Li, J. Mol. Cancer Ther. 2013, 12, 2874. (e) Han, X.; Zhang, X.; Li, H.; Huang, S.; Zhang, S.; Wang, F.; Shi, Y. Oncotarget 2015, 6, 38912. (f) Nami, B.; Donmez, H.; Kocak, N. Exp. Toxicol. Pathol. 2016, 68, 419. (g) Guha, P.; Kaptan, E.; Gade, P.; Kalvakolanu, D. Oncotarget 2017, 8, 68191. (h) Akiyama, T.; Oishi, K.; Wullaert, A. PLoS One 2016, 11, No. e0162448. (i) Yoshida, H.; Matsui, T.; Yamamoto, A.; Okada, T.; Mori, K. Cell 2001, 107, 881. (j) Urano, F.; Wang, X.; Bertolotti, A.; Zhang, Y.; Chung, P.; Harding, H.; Ron, D. Science 2000, 287, 664. (k) Hitomi, J.; Katayama, T.; Taniguchi, M.; Honda, A.; Imaizumi, K.; Tohyama, M. Neurosci. Lett. 2004, 357, 127. (l) Bravo, R.; Vicencio, J. M.; Parra, V.; Troncoso, R.; Munoz, J. P.; Bui, M.; Quiroga, C.; Rodriguez, A. E.; Verdejo, H. E.; Ferreira, J.; Iglewski, M.; Chiong, M.; Simmen, T.; Zorzano, A.; Hill, J. A.; Rothermel, B. A.; Szabadkai, G.; Lavandero, S. J. J. Cell Sci. 2011, 124, 2143. (3) (a) McDonald, L. A.; Barbieri, L. R.; Carter, G. T.; Lenoy, E.; Lotvin, J.; Petersen, P. J.; Siegel, M. M.; Singh, G.; Williamson, R. T. J. Am. Chem. Soc. 2002, 124, 10260. (b) Cheng, L.; Chen, W.; Zhai, L.; Xu, D.; Huang, T.; Lin, S.; Zhou, X.; Deng, Z. Mol. BioSyst. 2011, 7, 920. (c) Xu, D.; Liu, G.; Cheng, L.; Lu, X.; Chen, W.; Deng, Z. PLoS One 2013, 8, No. e76068. (4) Ochi, K.; Ezaki, M.; Iwani, M.; Komori, T.; Kohsaka, M. Eur. Patent Appl. 0333177 A2, 1989. (5) Lin, Y.-I.; Li, Z.; Francisco, G. D.; McDonald, L. A.; Davis, R. A.; Singh, G.; Yang, Y.; Mansour, T. S. Bioorg. Med. Chem. Lett. 2002, 12, 2341. (6) (a) Wang, Q.; Wang, Y.; Kurosu, M. Org. Lett. 2012, 14, 3372. (b) Wang, Y.; Aleiwi, B. A.; Wang, Q.; Kurosu, M. Org. Lett. 2012, 14, 4910. (7) Isolation of 3; see Supporting Information. (8) The mon-Boc-protected molecule 7 and di-Boc-protected molecule 8 were synthesized via total chemical syntheses according to the procedure previously reported; see Supporting Information. (9) (a) Nonoyama, K.; Ojima, H.; Nonoyama, M. Inorg. Chim. Acta 1976, 20, 127. (b) Choi, K.-Y.; Kim, Y.-S.; Choo, G. H.; Kim, J. G.; Suh, I. H. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 2001, 57, 1014. (10) Dubeau, M.-P.; Ghinet, M. G.; Jacques, P-É .; Clermont, N.; Beaulieu, C.; Brzezinski, R. Appl. Environ. Microbiol. 2009, 75, 1211. D
DOI: 10.1021/acs.orglett.8b03716 Org. Lett. XXXX, XXX, XXX−XXX
